Recombinant Human Transmembrane protein 235 (TMEM235)

What is TMEM235 and where is it primarily expressed in human tissues?

TMEM235 (Transmembrane protein 235) is a long noncoding RNA located on chromosome 10, downstream of BIRC5, and shares the same transcriptional direction. The full length of the transcript is 2853 nucleotides comprising seven exons. Despite its name suggesting a transmembrane protein, research indicates that TMEM235 does not encode a protein but functions as a regulatory RNA molecule. RNA-FISH analysis has shown that TMEM235 is primarily distributed in the cytoplasm of bone marrow mesenchymal stem cells (BMSCs) . The gene appears to have significant expression in bone marrow and neural tissues, with emerging research indicating potential roles in both bone regeneration and neural tissue contexts.

What is the molecular function of TMEM235 and through which pathways does it exert its effects?

TMEM235 functions as a competitive endogenous RNA (ceRNA) that binds to miR-34a-3p, preventing this microRNA from silencing BIRC5 mRNA expression. By competitively binding to miR-34a-3p, TMEM235 releases the inhibitory effect on BIRC5 (an inhibitor of apoptosis), thereby promoting cell survival under hypoxic conditions. The mechanism involves:

• Competitive binding to miR-34a-3p, which has the same binding site on both TMEM235 and BIRC5 mRNA

• Reduction of BIRC5 mRNA enrichment in miRNPs (microRNA ribonucleoprotein complexes)

• Increased expression of BIRC5 protein

• Inhibition of CASP-3 and CASP-9 activities

• Prevention of hypoxia-induced apoptosis

This miR-34a-3p/BIRC5 regulatory axis represents a critical pathway through which TMEM235 modulates cellular responses to hypoxic stress.

What are the recommended protocols for overexpressing or silencing TMEM235 in cell culture systems?

For effective manipulation of TMEM235 expression in experimental settings:

Overexpression protocol:

• Clone the full-length TMEM235 transcript (2853 nt) into an appropriate expression vector (e.g., pcDNA4/myc-his plasmid used in published research)

• Validate lack of protein-coding ability through immunoblotting with anti-myc antibody

• For stable overexpression, package the expression construct into a lentiviral vector (Lv-Lnc TMEM235)

• Transfect target cells (e.g., BMSCs) at appropriate MOI and select for stable integration

• Confirm overexpression through qPCR analysis comparing to control vectors (e.g., Lv-EGFP)

Silencing protocol:

• Design short hairpin RNAs (shRNAs) targeting conserved regions of TMEM235

• Package shRNAs into lentiviral vectors (Lv-Sh-Lnc TMEM235)

• Transfect target cells and select for stable integration

• Validate knockdown efficiency via qPCR, aiming for >70% reduction in expression

• Use scrambled shRNA sequences as negative controls

For experiments investigating the miR-34a-3p/BIRC5 axis, concurrent manipulation of miR-34a-3p (using Lv-miR-34a-3p) and BIRC5 (using Lv-BIRC5 or BIRC5 interference lentivirus) may be necessary to fully characterize the regulatory network.

How can researchers effectively measure TMEM235 expression levels in different experimental systems?

Quantifying TMEM235 expression accurately requires multiple complementary approaches:

RNA quantification:

• RT-qPCR using specific primers targeting unique regions of TMEM235 transcript

• Normalization to appropriate housekeeping genes (GAPDH, β-actin)

• RNA-seq for genome-wide expression analysis and identification of co-regulated genes

Subcellular localization:

• RNA fluorescence in situ hybridization (RNA-FISH) to visualize subcellular distribution

• Use 18S and U6 as positive controls for cytoplasmic and nuclear localization, respectively

• Confocal microscopy for high-resolution imaging of cellular distribution patterns

Functional association:

• RNA immunoprecipitation (RIP) assays to detect TMEM235 enrichment in miRNPs

• Dual-luciferase reporter assays to validate direct interactions with miRNA targets

• RNA pull-down assays to identify protein-binding partners

For clinical samples, microdissection techniques may be necessary to isolate specific cell populations before RNA extraction to avoid dilution of signal from non-expressing cells.

How does TMEM235 contribute to bone marrow mesenchymal stem cell (BMSC) survival in hypoxic conditions?

TMEM235 plays a crucial role in protecting BMSCs from hypoxia-induced apoptosis through several mechanisms:

• Under hypoxic conditions (0% O₂, 95% N₂, and 5% CO₂), TMEM235 expression is naturally downregulated, coinciding with increased apoptosis markers (Bax) and decreased anti-apoptotic factors (Bcl-2)

• Experimental overexpression of TMEM235 reverses these effects by:

  • Increasing Bcl-2 expression

  • Decreasing Bax and CASP-3 expression

  • Reducing apoptotic rate from >70% to significantly lower levels

• TMEM235 competitively binds to miR-34a-3p, preventing it from silencing BIRC5 expression

• Increased BIRC5 directly inhibits CASP-3 and CASP-9 activities, preventing the execution of apoptosis

• The anti-apoptotic effect is abolished when BIRC5 is downregulated, confirming the dependency on the TMEM235/miR-34a-3p/BIRC5 axis

This protective mechanism is particularly significant in the context of BMSC-based therapies for conditions like steroid-induced osteonecrosis of the femoral head (SONFH), where the hypoxic microenvironment of osteonecrotic areas typically leads to poor survival of transplanted cells.

What evidence supports the therapeutic potential of TMEM235-modified BMSCs for steroid-induced osteonecrosis of the femoral head (SONFH)?

In vivo studies have demonstrated significant therapeutic advantages of TMEM235-overexpressing BMSCs for SONFH treatment:

• In animal models of early SONFH, TMEM235-overexpressing BMSCs co-cultured with xenogeneic antigen-extracted cancellous bone (XACB) showed:

  • Higher fluorescence intensity of DiR-labeled cells, indicating improved survival

  • Increased expression of GFP and BIRC5

  • Significantly decreased proportion of TUNEL-positive (apoptotic) cells

  • Enhanced therapeutic efficacy compared to control BMSCs

• Conversely, silencing TMEM235 in BMSCs resulted in:

  • Lower DiR fluorescence intensity

  • Decreased GFP and BIRC5 expression levels

  • Increased proportion of TUNEL-positive cells

These findings suggest that genetic modification of BMSCs to overexpress TMEM235 could significantly enhance their therapeutic efficacy in SONFH by improving survival in the hypoxic microenvironment of osteonecrotic areas. This approach represents a promising strategy to overcome a major limitation of current BMSC-based therapies for SONFH.

How can researchers effectively validate the competitive endogenous RNA (ceRNA) function of TMEM235?

Validating TMEM235 as a ceRNA requires comprehensive experimental evidence:

Bioinformatic prediction and validation:

• Use established tools (miRDB, RNAhybrid) to predict binding sites for miRNAs on both TMEM235 and potential target mRNAs (e.g., BIRC5)

• Confirm sequence complementarity and binding energy calculations

• Design wild-type and mutant constructs with altered binding sites for functional validation

Direct binding experiments:

• RNA immunoprecipitation (RIP) assays:

  • Transfect cells with Lv-miR-34a-3p to upregulate miR-34a-3p

  • Perform RIP to isolate miRNPs

  • Quantify enrichment of TMEM235 and target mRNAs (BIRC5) in immunoprecipitates

  • Compare enrichment patterns in conditions with varied expression of TMEM235

• Luciferase reporter assays:

  • Clone predicted binding sites from TMEM235 and BIRC5 3'UTR into reporter constructs

  • Measure luciferase activity in the presence of miR-34a-3p mimics or inhibitors

  • Include mutated binding site constructs as controls

  • Demonstrate competitive effects by co-transfection experiments

Functional rescue experiments:

• Establish a system with manipulated expression of all three components (TMEM235, miR-34a-3p, and BIRC5)

• Demonstrate that phenotypes caused by miR-34a-3p can be rescued by TMEM235 overexpression

• Show that this rescue depends on BIRC5 expression

• Confirm with appropriate controls for each component

These approaches collectively provide strong evidence for ceRNA function and elucidate the specific molecular interactions involved.

What are the technical challenges in studying TMEM235 in different experimental models and how can they be addressed?

Researchers face several challenges when investigating TMEM235:

RNA stability and detection issues:

• Challenge: lncRNAs like TMEM235 often have lower expression levels than protein-coding genes
  Solution: Use specialized RNA extraction protocols optimized for lncRNAs; employ sensitive detection methods like droplet digital PCR

• Challenge: Degradation during sample processing
  Solution: Include RNase inhibitors during extraction; use fresh samples when possible; develop validated protocols for fixed tissues

Functional characterization:

• Challenge: Distinguishing direct vs. indirect effects
  Solution: Design rescue experiments with combinations of overexpression and knockdown of TMEM235, miR-34a-3p, and BIRC5; use binding site mutants to disrupt specific interactions

• Challenge: Translating in vitro findings to in vivo contexts
  Solution: Develop appropriate animal models; use tissue-specific expression systems; validate with human clinical samples when available

Evolutionary conservation:

• Challenge: Limited conservation of lncRNAs across species
  Solution: Focus on functional conservation rather than sequence conservation; compare binding site architecture between species; perform comparative studies in multiple model systems

Technical applications:

• Challenge: Efficient delivery of TMEM235 constructs
  Solution: Optimize viral packaging; test different promoters for expression; consider tissue-specific promoters for targeted expression

• Challenge: Monitoring expression in vivo
  Solution: Use reporter genes (GFP/luciferase); employ advanced imaging techniques; develop antibodies against BIRC5 to monitor downstream effects

Addressing these challenges requires multidisciplinary approaches and careful experimental design to ensure robust and reproducible findings.

What is the evidence for TMEM235 involvement in glioblastoma and other cancers?

While research on TMEM235 in cancer is emerging, preliminary evidence suggests potential roles:

• TMEM230, a related transmembrane protein, has been identified as a potential target for glioblastoma therapy, suggesting similar transmembrane proteins may have relevance in brain tumor biology

• The regulatory relationship between TMEM235 and the anti-apoptotic protein BIRC5 (survivin) is particularly noteworthy, as BIRC5:

  • Is frequently overexpressed in various cancers

  • Correlates with aggressive disease and poor prognosis

  • Represents a therapeutic target in multiple cancer types

• Analysis of TCGA data has been used to examine glioblastoma multiforme (GBM) and low-grade gliomas, suggesting a potential for examining TMEM235 expression patterns in these datasets

• The ability of TMEM235 to modulate cell survival under hypoxic conditions could be relevant to cancer biology, as hypoxic microenvironments are common features of solid tumors

Further research is needed to definitively establish the role of TMEM235 in cancer, including comprehensive expression analyses across tumor types, correlation with clinical outcomes, and functional studies in cancer cell lines and animal models.

How can TMEM235 be effectively targeted for therapeutic applications in bone regeneration?

Developing TMEM235-based therapeutic strategies for bone regeneration requires consideration of several approaches:

Genetic modification of BMSCs:

• Ex vivo transduction of patient-derived BMSCs with TMEM235 overexpression constructs

• Selection and expansion of modified cells

• Combination with appropriate scaffolds (e.g., xenogeneic antigen-extracted cancellous bone)

• Transplantation into affected areas

Development of miRNA inhibitors:

• Design of antagomirs targeting miR-34a-3p to mimic the effect of TMEM235 overexpression

• Development of delivery systems for local administration to bone defects

• Optimization of dose and timing to maximize BIRC5 expression and cell survival

Small molecule approaches:

• High-throughput screening for compounds that upregulate endogenous TMEM235 expression

• Identification of molecules that stabilize TMEM235 RNA or enhance its binding to miR-34a-3p

• Development of BIRC5 activators as downstream effectors of the TMEM235 pathway

Combination strategies:

• Integration with other bone regeneration approaches (e.g., growth factors, biomaterials)

• Sequential or simultaneous targeting of multiple components of the hypoxia response pathway

• Patient stratification based on disease severity and individual molecular profiles

For clinical translation, key considerations include safety assessment, optimization of delivery methods, determination of effective dosages, and development of appropriate outcome measures to assess therapeutic efficacy in human patients with conditions like SONFH.

What commercial reagents and tools are available for studying TMEM235 in laboratory settings?

Researchers have access to several commercial products and resources for TMEM235 studies:

Recombinant proteins and antibodies:

• Invitrogen Human TMEM235 Control Fragment Recombinant Protein (RP101769) - A recombinant protein with His-ABP-tag that can be used for blocking experiments with corresponding antibody PA5-62878

• The protein sequence reported is "SDYWYILEVADAGNGSAWPGRAELLSSHPGLWRICEVL" and is produced in E. coli with >80% purity by SDS-PAGE

• The protein is supplied in 1 M urea, PBS without preservative at pH 7.4, with concentration ≥5.0 mg/mL

Experimental applications:

• For IHC/ICC and Western blotting experiments, a 100x molar excess of the protein fragment control is recommended

• Pre-incubation of the antibody-protein control fragment mixture for 30 minutes at room temperature is suggested to ensure effective blocking

Storage and handling:

• The recombinant protein should be stored at -20°C and freeze/thaw cycles should be avoided

• The protein is supplied in liquid form without preservatives, requiring careful handling to prevent contamination

When designing experiments, researchers should consider that this control fragment represents only a portion of the full TMEM235 sequence and may be most appropriate for validating antibody specificity rather than functional studies of the complete lncRNA.

What bioinformatic resources and databases are most useful for analyzing TMEM235 expression and interaction networks?

Several computational resources can facilitate TMEM235 research:

Expression databases:

• The Cancer Genome Atlas (TCGA) - Contains mRNAseq datasets that can be analyzed for TMEM235 expression across various cancers including glioblastoma multiforme (GBM) and low-grade gliomas (LGG)

• GTEx (Genotype-Tissue Expression) project - Provides expression data across normal human tissues

• Human Protein Atlas - Offers protein expression data that may include related proteins

Analysis tools:

• R package TCGA2STAT - Used for analyzing TCGA data, as demonstrated in TMEM230 studies

• RSEM (RNA-Seq by Expectation Maximization) - A method for normalizing RNA-seq data

• DESEQ2 - Used for differential gene expression analysis with appropriate p-value cutoffs (e.g., <0.0001) and log2 fold change thresholds (e.g., >1)

miRNA prediction tools:

• miRDB - Used to predict binding sites for miRNAs on TMEM235

• RNAhybrid - Applied to predict interactions between TMEM235 and miRNAs

• ENCORI (starBase) - Can be used to explore RNA-RNA and RNA-protein interactions

Pathway analysis:

• Ingenuity Pathway Analysis (IPA) - For interpreting molecular data in the context of biological systems

• STRING database - For protein-protein interaction networks relevant to BIRC5 and related factors

• Reactome - For pathway enrichment analysis of genes co-regulated with TMEM235

These resources collectively enable comprehensive analysis of TMEM235's expression patterns, potential regulatory interactions, and functional implications across different biological contexts and disease states.

What are the most promising research directions for elucidating TMEM235's broader roles beyond bone regeneration?

Several emerging areas warrant investigation to fully understand TMEM235's biological significance:

Neurological contexts:

• Given the apparent role of related transmembrane proteins in glioblastoma, investigation of TMEM235 in neural development and neurological disorders

• Examination of potential neuroprotective effects against hypoxic damage in stroke or neurodegenerative conditions

• Analysis of expression patterns across neural cell types and brain regions

Other hypoxia-sensitive tissues:

• Cardiac tissue - Exploring potential cardioprotective effects following ischemic injury

• Retinal tissue - Investigating roles in conditions like diabetic retinopathy

• Wound healing - Examining potential to enhance tissue repair in compromised vascular environments

Broader regulatory networks:

• Comprehensive identification of other miRNAs that may interact with TMEM235

• Exploration of additional mRNA targets that might be regulated by the same mechanism

• Investigation of potential protein-binding partners that could influence TMEM235 stability or function

Evolutionary aspects:

• Comparative analysis of TMEM235 conservation and function across species

• Investigation of tissue-specific expression patterns in different model organisms

• Examination of potential specialized roles in human biology versus other mammals

These research directions could significantly expand our understanding of TMEM235's biological importance beyond its established role in bone marrow stem cell survival under hypoxic conditions.

What methodological advances would enhance research capabilities for studying TMEM235 and similar lncRNAs?

Advancing TMEM235 research would benefit from several methodological innovations:

Improved detection and visualization:

• Development of high-sensitivity in situ hybridization methods for low-abundance lncRNAs

• Application of single-molecule RNA FISH techniques for precise subcellular localization

• Creation of reporter constructs that accurately reflect endogenous expression patterns

Functional interrogation:

• CRISPR-Cas13 systems for precise RNA targeting and manipulation

• Advanced RNA structure probing techniques (SHAPE-seq, icSHAPE) to understand structural determinants of TMEM235 function

• High-throughput methods to identify RNA-protein interactions (RNA-protein interaction detection, RAPID)

In vivo modeling:

• Development of transgenic animal models with inducible, tissue-specific TMEM235 expression

• Application of AAV-mediated delivery systems for in vivo modulation of TMEM235 levels

• Advanced imaging techniques to monitor real-time dynamics of TMEM235 in living systems

Translational approaches:

• Development of RNA-targeting therapeutics (antisense oligonucleotides, small molecules)

• Optimization of RNA delivery systems (lipid nanoparticles, exosomes) for clinical applications

• Identification of biomarkers that reflect TMEM235 pathway activation for patient stratification

These methodological advances would not only benefit TMEM235 research specifically but would also contribute to the broader field of lncRNA biology and therapeutic development.

What are the optimal experimental conditions for studying TMEM235 function in hypoxic environments?

Precise experimental parameters are crucial for reproducible TMEM235 research in hypoxic conditions:

Hypoxia induction protocol:

• Gas composition: 0% O₂, 95% N₂, and 5% CO₂

• Duration: 48 hours for optimal observation of hypoxia-induced effects

• Temperature: Maintained at 37°C throughout the experiment

• Humidity: >95% to prevent medium evaporation

• Control conditions: Normoxia (21% O₂, 5% CO₂, 74% N₂)

Cell culture considerations:

• Cell density: 2-3 × 10⁵ cells/cm² for BMSCs to ensure appropriate cell-cell communication

• Medium composition: Standard growth medium supplemented with 10% FBS, potentially pre-equilibrated to hypoxic conditions

• Serum reduction: Consider gradually reducing serum concentration to minimize proliferation and focus on survival effects

Analysis timepoints:

• Early response: 6-12 hours for transcriptional changes

• Intermediate response: 24 hours for protein expression changes

• Late response: 48 hours for apoptosis assessment

• Extended observation: 72 hours for long-term survival evaluation

Readout parameters:

• Apoptosis: Flow cytometry with Annexin V/PI staining

• Cell viability: MTT or WST-1 assays

• Gene expression: qPCR for TMEM235, BIRC5, miR-34a-3p

• Protein markers: Western blotting for Bcl-2, Bax, CASP-3, CASP-9

• Enzyme activity: Fluorometric assays for CASP-3 and CASP-9 activities

These standardized conditions enable meaningful comparison between experiments and facilitate translation of in vitro findings to in vivo applications.

What quantitative data should researchers track to fully characterize TMEM235's effects in experimental systems?

To comprehensively evaluate TMEM235's impact, researchers should collect the following quantitative measurements:

Expression parameters:

Functional readouts:

Molecular interactions:

In vivo parameters:

These quantitative parameters provide a comprehensive profile of TMEM235's molecular and functional effects, enabling robust characterization across different experimental conditions and model systems.